Expression Cloning of a Receptor for C5a Anaphylatoxin on Differentiated HL-60 Cells

Biochemistry. 1991 Mar 26;30(12):2993-9. doi: 10.1021/bi00226a002.

Abstract

A cDNA clone encoding the human C5a anaphylatoxin receptor has been isolated by expression cloning from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with dibutyryladenosine cyclic monophosphate. The cDNA clone was able to transfer to COS-7 cells the capacity to specifically bind iodinated human recombinant C5a. The cDNA was 2.3 kb long, with an open reading frame encoding a 350-residue polypeptide. Cross-linking of iodinated C5a to the plasma membrane of transfected COS cells revealed a complex with an apparent molecular mass of 52-55 kDa, similar to that observed for the constitutively expressed receptor in differentiated HL-60 cells or human neutrophils. Although differentiated HL-60 cells display a single class of binding sites, with a dissociation constant of approximately 800-900 pM, the C5a-R cDNA, expressed in COS cells, generates both high-affinity (1.7 nM) and low-affinity (20-25 nM) receptors. Sequence comparison established that the degree of sequence identity between the C5a receptor and the N-formylpeptide receptor is 34%.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anaphylatoxins / genetics*
  • Animals
  • Blotting, Northern
  • Cell Differentiation
  • Cell Line
  • Cloning, Molecular
  • Complement C5a / genetics*
  • Cross-Linking Reagents
  • DNA / genetics
  • Dogs
  • Gene Expression Regulation
  • Humans
  • Molecular Sequence Data
  • RNA, Messenger / analysis
  • RNA, Messenger / genetics
  • Radioligand Assay
  • Receptor, Anaphylatoxin C5a
  • Receptors, Complement / genetics
  • Receptors, Complement / metabolism*
  • Sequence Alignment
  • Sequence Homology, Nucleic Acid
  • Transfection

Substances

  • Anaphylatoxins
  • Cross-Linking Reagents
  • RNA, Messenger
  • Receptor, Anaphylatoxin C5a
  • Receptors, Complement
  • Complement C5a
  • DNA

Associated data

  • GENBANK/J05327
  • GENBANK/M60676
  • GENBANK/M62347
  • GENBANK/M62348
  • GENBANK/M62349
  • GENBANK/M62350
  • GENBANK/M62351
  • GENBANK/M62352
  • GENBANK/M62353
  • GENBANK/M62354
  • GENBANK/M62505